Cytotoxic Activity of Cyclamen Persicum Ethanolic Extract on MCF-7, PC-3 and LNCaP Cancer Cell Lines

It is important to develop new approaches to increase the efficacy of cancer treatments. Nowadays, the uses of natural products to treat cancer are very common. In addition, working with plants that are endemic to Palestine and determining the biological activities of these plant extracts, is extremely important due to the potential for new drug development. Cyclamen persicum is used in traditional medicinal to treat anti-rheumatic, diarrhea, abdominal pains, edema, abscesses, eczema, cancer and other ailments. In this study the cytotoxic effect of C. persicum tubers and leaves ethanolic extracts were studied against MCF-7, PC-3 and LNCaP cancer cell lines, using mitochondrial dehydrogenase enzyme method. Results showed the remarkable cytotoxic activity of C. persicum extracts, against breast and prostate adenocarcinoma. For tubers extract the IC50 value was found to be 0.05 mg/ml for the three cell lines. Although the leaves extract the IC50 value was found to be 0.25 mg/ml for PC-3 and MCF-7 cell lines, while LNCaP cell inhibition were less than 30% at all tested leaves extract concentrations. MCF-7 cells exhibited the highest sensitivity to the C. persicum extracts, compared to PC-3 and LNCaP cell lines evaluated. In contrast, LNCaP cells generally exhibited the lowest sensitivity to extracts. These results displayed that C. persicum is a good source for natural products with antitumor compounds that can be further exploited for the development of a potential therapeutic anticancer agent.


Materials and methods 2.1 Plant Collection
Tubers and leaves of C. persicum were collected between March and July from Nablus mountains (West Bank, Palestine) (altitude 600-900 m). Authentification of the plants was conducted by Prof. Dr. Ali-Shtayeh by comparison with plant specimens located at BERC Herbarium and voucher specimens were deposited in the Biodiversity and Biotechnology Research Unit at BERC Herbarium, Til, Nablus, Palestine.

Preparation of crude extracts
Tubers and leaves of C. persicum (100 g) dried in the shade at room temperature, powdered and infused in 70% ethanol (1:5 w/v) at room temperature with periodic shaking for 72h. The filtrates were condensed by evaporating the solvents using rotary evaporator, dried under reduced pressure, and stock solutions of 10 mg/ml in dimethyl sulphoxide (DMSO) were prepared at room temperature and stored at -20°C. (Ali-Shtayeh et al., 2013).

Cytotoxicity
Cells were seeded in 96 well-plates at a density of 4x10 4 cell/ 200µl for LNCaP cells, while 3x10 4 cells/ 200µl for PC-3 and MCF-7cell lines. The cells were allowed to attach in a 5% CO2 incubator at 37° C for 24 h. In order to prepare the 4 different doses of extracts (0.05, 0.125, 0.25 and 0.5 mg/ml), crude extract was serially diluted into supplemented media using a separate 96-well plate and extract doses applied to the cells. After incubation for 24 hours viable cells were quantitated by using 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (MERCK, Germany) assay. In brief after cells incubation for 24 hours, medium was aspirated carefully and discarded. After that 50 µl of MTT solution (1.5mg/ml) was added to each well and incubated for 3 h, then100 µl of iced acetone-ethanol solution (1:1) were added to dissolve violet crystals. Viability was quantitated by using a 96 well-plate reader at 570 nm wavelength, with a reference wave length of 650 nm. The percentage of cell survival was determined as (mean of treated wells/ mean of untreated control wells) x100%. DMSO was used as a negative control (Srivastava et al., 1998;Ozcan et al., 2016).

Statistical analysis
IC50 (50% growth inhibition) values were defined as the concentration of the extract where there is a 50% loss of total metabolic activity as compared to untreated controls and are reported as mean ± standard deviation (SD). The data were analyzed and the treatment were compared using analysis of variance (ANOVA) obtained by Duncan's multiple-range test and P values less than 0.05 were considered to be significant. All experiments have been conducted in three replicates.

Results
In order to evaluate the cytotoxic effect of ethanolic extracts from C. persicum (tubers and leaves), MTT assay with three different cancer cell lines (PC3, LNCaP, and MCF-7) were performed. To determine the IC50 value the extracts were screened for its cytotoxicity at different concentrations (0.05, 0.125, 0.25 and 0.5 mg/ml). When cells treated with ethanolic extract of the tubers and leaves of C. persicum, there was a concentration dependent cytotoxic effect. As the concentration increased from 0.05-0.5 mg, percentage of inhibition increases ( Table 1). 29.3 ± 3.6 28.2 ± 7.4 22.6 ± 5.7 0 (Control) 0 0 0 This study showed the remarkable cytotoxic activity of C. persicum extracts, against breast and prostate adenocarcinoma. For tubers extract the IC50 value was found to be 0.05 mg/ml for the three cell lines. While the leaves extract the IC50 value was found to be 0.25 mg/ml for PC-3 and MCF-7 cell lines, but LNCaP cell inhibition were less than 30% at all tested leaves extract concentrations (Table 1. & Figure 1.). MCF-7 cells exhibited the highest sensitivity to the C. persicum extracts, with lower IC50 values as compared to PC-3 and LNCaP cell lines evaluated. In contrast, LNCaP cells generally exhibited the lowest sensitivity to the plant extracts (Table 1).
A B Figure 1. % Cell viability of PC3, LNCaP, and MCF-7 cell lines after 24 hours incubation with ethanolic extracts prepared from C. persicum tubers (A) and leaves (B). Cell viability was determined using MTT assay. Results are expressed as mean ± S.D (N= 2). Figure 1 shows that a significant decrease in cell viability with 0.05, 0.125, 0.25 and 0.5 mg/ml from C. persicum tuber extracts after 24 h., and all doses showed viability by 10% at 0.5 mg/ml when compared with the control group. Cell viability decreased with increasing of leaves extracts concentration and all doses showed cell viability less than 30% at 0.5 mg/ml extract concentration for PC-3, MCF-7 cell lines. While LNCaP cell viability were more than 70% with 0.05, 0.125, 0.25 and 0.5 mg/ml from leave extracts after 24 hours compared with the control group (Table 1 & Figure 1).
C. persicum tubers extract appeared to be the most effective extract compared to the leaves extract applied to the PC-3, MCF-7 and LNCaP cells. It can be seen that LNCaP cells were more resistant to the treatment with C. persicum tubers extract compared with other cell lines.

Discussion
Most of the currently used anticancer drugs are highly toxic, expensive, and resistance mechanisms pose a significant problem (Hait and Hambley, 2009;Alzeer et al., 2014). There is a continuing need to identify new drug candidates that are more effective, widely available and less toxic. Plants extracts are an important source of potentially useful compounds for the development of new anticancer drugs.
In our study we investigated ethanolic extraction of C. persicum Palestinian medicinal plant for cytotoxic activities against breast and prostate adenocarcinoma cell lines. Results reported that C. persicum showed strong cytotoxic activity against PC-3, MCF-7 and LNCaP cell lines with an inhibition of > 90% at 0.5 mg/ml (Table 1). C. persicum is currently used for anticancer treatments in traditional Palestinian medicine (Khalilia, 2001;Ali-Shtayeh and Jamous, 2008;Ali-Shtayeh et al., 2011). The anti-oxidant properties of this species have been previously evaluated, which suggested the possibility of anti-proliferative potential for these plants (Metın et al. 2013;Mazouz andDjeddi, 2014 andJaradat et al. 2015). Although the cytotoxicity of C. persicum has been previously reported on breast, colon, hepatocellular carcinoma, lung, pancreatic and cervical cancer cell lines (Yildiz et al., 2013;El Hosry et al., 2014). Therefore, this plant extracts with significant cytotoxic activity should be further assessed using animal models.
Results of this study shows the inhibitory effect against the three cell lines varied significantly between the two C. persicum extracts. At concentration of 0.5 mg/ml, tubers extract was the most toxic (> 90% inhibition). While leaves extract showed inhibition effect less than 70%. Cyclamen spp (Primulaceae) are rich in toxic saponins, known to have interesting biological activities (Spoerke et al., 1987;Çaliş et al., 1997;Altunkeyik et al., 2012;Sajjadi et al., 2016;Stanojević et al., 2018). In a study conducted by El Hosry et al., 2014, showed the remarkable cytotoxic activity of saxifragifolin B (saponin derived from cyclamen species), against breast adenocarcinoma and lung carcinoma and its chemo protective activity against mitomycin C.
From our results, MCF-7 cells exhibited the highest sensitivity to the C. persicum extracts, with lower IC50 values as compared to PC-3 and LNCaP cell lines evaluated. These results are agreement with previous studies on various plant extracts (Yaacob et al., 2010;Subarnas et al., 2012). In order to identify the bioactive component(s) and to further understand the mechanism of action of C. persicum extracts should be evaluated.

Conclusion
The results of the present study demonstrated that the extracts of C. persicum tubers and leaves possessed strong cytotoxic activity and can be easily accessible source of natural anticancer products in cell cultures. C. persicum tubers extract appeared to be more effective compared to the leaves extract applied to the PC-3, MCF-7 and LNCaP cells. These results justified the use of C. persicum in traditional medicine.
The researcher decaled that the components responsible for antitumor activity of the extracts of C. persicum are unclear. Future studies will be aimed at investigating the effects of different parts of C. persicum upon isolating and identifying the substances responsible for the anticancer effects of the solvent extracts. We hope that the results of this study will play an important role in designing and developing natural anticancer drugs and will make contributions to other studies that might be conducted in this area.